A fundamental biological question is how external stimuli evoke cellular responses. One prevalent type of environmental sensor in prokaryotic organisms is the two-component system (TCS). A canonical TCS consists of a membrane-spanning sensor histidine kinase (SHK) and a cytoplasmic response regulator (RR). A recent survey of 145 prokaryotic genomes detected more than 4000 TCSs. TCSs regulate a diverse array of virulence factors. Identifying two-component signaling pathways that lead to enhanced pathogenicity is essential to understanding complex host-pathogen interactions. The specific hypothesis examined is that SHK-membrane interactions can be identified and harnessed to modulate SHK signal output in a predictable manner. Specifically targeting individual SHK signal output could be used to rapidly unravel two-component signal pathways in any pathogenic organism of interest. Three specific aims are designed to develop this methodology. Specific Aim 1 will identify and harness interactions between TM2 of two well-characterized SHKs, EnvZ and NarX, and the cellular membrane using fluorescent and enzymatic reporters. Specific Aim 2 will determine the role of protein-membrane interactions during signal transduction by the HAMP domains of EnvZ and NarX. These interactions will be examined through use of random mutagenesis and fluorescent microscopy. Specific Aim 3 will couple transcriptional profiling with harnessed SHK-membrane interactions (from Specific Aims 1 and 2) to establish a methodology for rapidly unraveling two-component signaling pathways in any pathogenic organism of interest. The long-term goal of this research is to create a method for rapidly identifying the signaling pathways that regulate the virulence of pathogenic microorganisms. This research will lead to a better understanding of complex host-pathogen interactions and will result in the detection of previously unidentified therapeutic targets.